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Jean-Marc Ghigo

Publications and source records attributed to Jean-Marc Ghigo.

15 recordsLinked to original sources
PubMed2006

Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide.

The development of surface-attached biofilm bacterial communities is considered an important source of nosocomial infections. Recently, bacterial interference via signaling molecules and surface active compounds was shown to antagonize biofilm formation, suggesting that nonantibiotic molecules produced during competitive interactions between bacteria could be used for biofilm reduction. Hence, a better understanding of commensal/pathogen interactions within bacterial community could lead to an improved control of exogenous pathogens. To reveal adhesion or growth-related bacterial interference, we investigated interactions between uropathogenic and commensal Escherichia coli in mixed in vitro biofilms. We demonstrate here that the uropathogenic strain CFT073 and all E. coli expressing group II capsules release into their environment a soluble polysaccharide that induces physicochemical surface alterations, which prevent biofilm formation by a wide range of Gram-positive and Gram-negative bacteria. We show that the treatment of abiotic surfaces with group II capsular polysaccharides drastically reduces both initial adhesion and biofilm development by important nosocomial pathogens. These findings identify capsular polymers as antiadhesion bacterial interference molecules, which may prove to be of significance in the design of new strategies to limit biofilm formation on medical in dwelling devices.

Bacterial Adhesion↗
PubMed2006

An IncI1 plasmid contributes to the adherence of the atypical enteroaggregative Escherichia coli strain C1096 to cultured cells and abiotic surfaces.

Enteroaggregative Escherichia coli (EAEC) is defined by a characteristic "stacked-brick" aggregative adherence (AA) pattern to cultured cells. In well-studied EAEC prototype strains (called typical EAEC strains), the AA phenotype requires aggregative adherence fimbriae (AAFs). However, previous studies suggest that known AAF alleles are not found in all EAEC strains. To define mechanisms contributing to adherence in an atypical strain, we studied EAEC strain C1096. An E. coli K12 derivative carrying two plasmids, designated pSERB1 and pSERB2, from C1096 adhered to cell lines and exhibited an AA pattern. Nucleotide sequence analysis of pSERB1 indicated that it is related to plasmids of the IncI1 incompatibility group. These plasmids encode genes involved in pilus-mediated conjugal transfer, as well as pilL-V, which encodes a second pilus of the type IV family. Insertional inactivation of the gene predicted to encode the major type IV pilin subunit (pilS) reduced conjugal transfer of the plasmid by 4 orders of magnitude. Adherence of the mutant strain to polystyrene and to HT29 cells was reduced by approximately 21% and 75%, respectively. In a continuous-flow microfermentor, the pilS inactivation reduced mature biofilm formation on a glass slide by approximately 50%. In addition, the simultaneous presence of both pSERB1 and pSERB2 plasmids promoted pilS-independent biofilm formation. We conclude that the IncI1 plasmid of EAEC C1096 encodes a type IV pilus that contributes to plasmid conjugation, epithelial cell adherence, and adherence to abiotic surfaces. We also observe that AA can be mediated by factors distinct from AAF adhesins.

Bacterial Adhesion↗
PubMed2006

The transcriptional antiterminator RfaH represses biofilm formation in Escherichia coli.

We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5Leu) on biofilm formation by uropathogenic Escherichia coli (UPEC) strain 536. Only inactivation of rfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation by E. coli 536. rfaH inactivation in nonpathogenic E. coli K-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and an rfaH mutant of this strain revealed that deletion of rfaH correlated with increased expression of flu orthologs. flu encodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion of rfaH leads to increased levels of flu and flu-like transcripts in E. coli K-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype of E. coli MG1655rfaH was reversed upon inactivation of flu. Deletion of the two flu orthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaH are mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to null rfaH-dependent lipopolysaccharide depletion. Although the roles of rfaH in the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.

Adhesins, Bacterial↗
PubMed2006

A CsgD-independent pathway for cellulose production and biofilm formation in Escherichia coli.

Bacterial growth on a surface often involves the production of a polysaccharide-rich extracellular matrix that provides structural support for the formation of biofilm communities. In Salmonella, cellulose is one of the major constituents of the biofilm matrix. Its production is regulated by CsgD and the diguanylate cyclase AdrA that activates cellulose synthesis at a posttranscriptional level. Here, we studied a collection of Escherichia coli isolates, and we found that the ability to produce cellulose is a common trait shared by more than 50% of the tested strains. We investigated the genetic determinants of cellulose production and its role in biofilm formation in the commensal strain E. coli 1094. By contrast with the Salmonella cellulose regulatory cascade, neither CsgD nor AdrA is required in E. coli 1094 to regulate cellulose production. In this strain, an alternative cellulose regulatory pathway is used, which involves the GGDEF domain protein, YedQ. Although AdrA(1094) is functional, it is weakly expressed in E. coli 1094 compared to YedQ, which constitutively activates cellulose production under all tested environmental conditions. The study of cellulose regulation in several other E. coli isolates showed that, besides the CsgD/AdrA regulatory pathway, both CsgD-independent/YedQ-dependent and CsgD-independent/YedQ-independent pathways are found, indicating that alternative cellulose pathways are common in E. coli and possibly in other cellulose-producing Enterobacteriaceae.

Amino Acid Motifs↗
PubMed2005

Finding gene-expression patterns in bacterial biofilms.

The production of biofilms by bacteria is a lifestyle that is thought to require or involve a differential gene expression compared with that of planktonic bacteria. Recently, we have witnessed a change of focus from the simple hunt for hypothetical essential biofilm genes to the identification of late and more complex biofilm functions. However, finding common bacterial biofilm gene-expression patterns through global expression analysis remains difficult. Owing to the apparently minimal overlap between functions involved in biofilm formation by different bacteria, exploring the biofilm lifestyle could prove to be a case-by-case task for which global approaches show their limits.

Adaptation, Physiological↗
PubMed2005

The Yak1p kinase controls expression of adhesins and biofilm formation in Candida glabrata in a Sir4p-dependent pathway.

Biofilm is the predominant type of microbial development in natural environments, and potentially represents a major form of resistance or source of recurrence during host infection. Although a large number of studies have focussed on the genetics of bacterial biofilm formation, very little is known about the genes involved in this type of growth in fungi. A genetic screen for Candida glabrata Biofilm mutants was performed using a 96-well plate model of biofilm formation. Study of the isolated mutant strains allowed the identification of four genes involved in biofilm formation (RIF1, SIR4, EPA6 and YAK1). Epa6p is a newly identified adhesin required for biofilm formation in this pathogenic yeast. EPA6 and its close paralogue EPA7 are located in subtelomeric regions and their transcription is regulated by Sir4p and Rif1p, two proteins involved in subtelomeric silencing. Biofilm growth conditions induce the transcription of EPA6 and EPA7: this is dependent on the presence of an intact subtelomeric silencing machinery and is independent of the Mpk1p signalling pathway. Finally, the kinase Yak1p is required for expression of both adhesin genes and acts through a subtelomeric silencing machinery-dependent pathway.

Biofilms↗
PubMed2005

BapA, a large secreted protein required for biofilm formation and host colonization of Salmonella enterica serovar Enteritidis.

In environmental settings, biofilms represent the common way of life of microorganisms. Salmonella enterica serovar Enteritidis, the most frequent cause of gastroenteritis in developed countries, produces a biofilm whose matrix is mainly composed of curli fimbriae and cellulose. In contrast to other bacterial biofilms, no proteinaceous compound has been reported to participate in the formation of this matrix. Here, we report the discovery of BapA, a large cell-surface protein required for biofilm formation by S. Enteritidis. Deletion of bapA caused the loss of the capacity to form a biofilm whereas the overexpression of a chromosomal copy of bapA increased the biofilm biomass formation. We provide evidence that overproduction of curli fimbriae and not cellulose can compensate for the biofilm deficiency of a bapA mutant strain. BapA is secreted through a type I protein secretion system (BapBCD) situated downstream of the bapA gene and was found to be loosely associated with the cell surface. Experiments with mixed bacterial populations positive or negative for BapA showed that BapA minus cells are not recruited into the biofilm matrix. The expression of bapA is coordinated with that of genes encoding curli fimbriae and cellulose, through the action of csgD. Studies on the contribution of BapA to S. Enteritidis pathogenesis revealed that orally inoculated animals with a bapA-deficient strain survived longer than those inoculated with the wild-type strain. Also, a bapA mutant strain showed a significantly lower colonization rate at the intestinal cell barrier and consequently a decreased efficiency for organ invasion compared with the wild-type strain. Taken together, these data demonstrate that BapA contributes both to biofilm formation and invasion through the regular Salmonella infection route.

Amino Acid Sequence↗
PubMed2005

Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins.

In bacteria, whereas disruption methods have been improved recently, the use of plasmid complementation strategies are still subject to limitations, such as cloning difficulties, nonphysiological levels of gene expression, or a requirement for antibiotics as plasmid selection pressure. Moreover, because of the pleiotropic modifications of cell physiology often induced by plasmid-based complementation, these strategies may introduce biases when biological process such as adhesion or biofilm formation are studied. We developed a plasmid-free approach that combines the lambda-red linear DNA recombination method with site-directed insertion of a repression and expression (RExBAD) cassette which places a functional pBAD promoter upstream of a target gene. We showed that this method permits both inactivation and modulation of most Escherichia coli gene expression, including expression of toxin and essential genes. We used this strategy to study adhesion and bacterial biofilms by placing the RExBAD cassette in front of the tra operon, encoding the DNA transfer and pilus genes on the F conjugative plasmid, and in front of flu, the antigen 43 (Ag43) autotransporter adhesin-encoding gene. In silico analysis revealed the existence of 10 genes with homology to the Ag43 gene that were good candidates for genes that encode putative new adhesins in E. coli. We used the RExBAD strategy to study these genes and demonstrated that induction of expression of four of them is associated with adhesion of E. coli to abiotic surfaces. The potential use of the RExBAD approach to study the function of cryptic or uncharacterized genes in large-scale postgenomic functional analyses is discussed.

Adhesins, Escherichia coli↗
PubMed2005

CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli.

Curli fibers could be described as a virulence factor able to confer adherence properties to both abiotic and eukaryotic surfaces. The ability to adapt rapidly to changing environmental conditions through signal transduction pathways is crucial for the growth and pathogenicity of bacteria. OmpR was shown to activate csgD expression, resulting in curli production. The CpxR regulator was shown to negatively affect curli gene expression when binding to its recognition site that overlaps the csgD OmpR-binding site. This study was undertaken to clarify how the interplay between the two regulatory proteins, OmpR and CpxR, can affect the transcription of the curli gene in response to variation of the medium osmolarity. Band-shift assays with purified CpxR proteins indicate that CpxR binds to the csgD promoter region at multiple sites that are ideally positioned to explain the csg repression activity of CpxR. To understand the physiological meaning of this in vitro molecular phenomenon, we analyzed the effects of an osmolarity shift on the two-component pathway CpxA/CpxR. We establish here that the Cpx pathway is activated at both transcriptional and posttranscriptional levels in response to a high osmolarity medium and that CpxR represses csgD expression in high-salt-content medium, resulting in low curli production. However, csgD repression in response to high sucrose content is not mediated by CpxR but by the global regulatory protein H-NS. Therefore, multiple systems (EnvZ/OmpR, Cpx, Rcs, and H-NS) appear to be involved in sensing environmental osmolarity, leading to sophisticated regulation of the curli genes.

Bacterial Proteins↗
PubMed2004

Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression.

The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K-12 biofilm. We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up- or downregulation by a factor greater than two, and that 10% of the E. coli genome is significantly differentially expressed. The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions. We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms. We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes. Using gene disruption of 54 of the most biofilm-induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological signature.

Biofilms↗
PubMed2004

Excision of the high-pathogenicity island of Yersinia pseudotuberculosis requires the combined actions of its cognate integrase and Hef, a new recombination directionality factor.

The Yersinia high-pathogenicity island (HPI) encodes the siderophore yersiniabactin-mediated iron uptake system. The HPI of Yersinia pseudotuberculosis I has previously been shown to be able to excise precisely from the bacterial chromosome by recombination between the attB-R and attB-L sites flanking the island. However, the nature of the Y. pseudotuberculosis HPI excision machinery remained unknown. We show here that, upon excision, the HPI forms an episomal circular molecule. The island thus has the ability to excise from the chromosome, circularize and reintegrate itself, either in the same location or in another asn tRNA copy. We also demonstrate that the HPI-encoded bacteriophage P4-like integrase (Int) plays a critical role in HPI excision and that, like phage integrases, it acts as a site-specific recombinase that catalyses both excision and integration reactions. However, Int alone cannot efficiently promote recombination between the attB-R and attB-L sites, and we demonstrate that a newly identified HPI-borne factor, designated Hef (for HPI excision factor) is also required for this activity. Hef belongs to a family of recombination directionality factors. Like the other members of this family, Hef probably plays an architectural rather than a catalytic role and promotes HPI excision from the chromosome by driving the function of Int towards an excisionase activity. The fact that the HPI, and probably several other pathogenicity islands, carry a machinery of integration/excision highly similar to those of bacteriophages argues for a phage-mediated acquisition and transfer of these elements.

Amino Acid Sequence↗
PubMed2004

Candida albicans biofilms: a developmental state associated with specific and stable gene expression patterns.

Like many bacteria, yeast species can form biofilms on several surfaces. Candida albicans colonizes the surfaces of catheters, prostheses, and epithelia, forming biofilms that are extremely resistant to antifungal drugs. We have used transcript profiling to investigate the specific properties of C. albicans biofilms. Biofilm and planktonic cultures produced under different conditions of nutrient flow, aerobiosis, or glucose concentration were compared by overall gene expression correlation. Correlation was much higher between biofilms than planktonic populations irrespective of the growth conditions, indicating that biofilm populations formed in different environments display very similar and specific transcript profiles. A first cluster of 325 differentially expressed genes was identified. In agreement with the overrepresentation of amino acid biosynthesis genes in this cluster, Gcn4p, a regulator of amino acid metabolism, was shown to be required for normal biofilm growth. To identify biofilm-related genes that are independent of mycelial development, we studied the transcriptome of biofilms produced by a wild-type, hypha-producing strain and a cph1/cph1 efg1/efg1 strain defective for hypha production. This analysis identified a cluster of 317 genes expressed independently of hypha formation, whereas 86 genes were dependent on mycelial development. Both sets revealed the activation of the sulfur-amino acid biosynthesis pathway as a feature of C. albicans biofilms.

Amino Acids, Sulfur↗
PubMed2003

SarA and not sigmaB is essential for biofilm development by Staphylococcus aureus.

Staphylococcus aureus biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA/PNAG), the product of the ica operon. The staphylococcal accessory regulator, SarA, is a central regulatory element that controls the production of S. aureus virulence factors. By screening a library of Tn917 insertions in a clinical S. aureus strain, we identified SarA as being essential for biofilm development. Non-polar mutations of sarA in four genetically unrelated S. aureus strains decreased PIA/PNAG production and completely impaired biofilm development, both in steady state and flow conditions via an agr-independent mechanism. Accordingly, real-time PCR showed that the mutation in the sarA gene resulted in downregulation of the ica operon transcription. We also demonstrated that complete deletion of sigmaB did not affect PIA/PNAG production and biofilm formation, although it slightly decreased ica operon transcription. Furthermore, the sarA-sigmaB double mutant showed a significant decrease of ica expression but an increase of PIA/PNAG production and biofilm formation compared to the sarA single mutant. We propose that SarA activates S. aureus development of biofilm by both enhancing the ica operon transcription and suppressing the transcription of either a protein involved in the turnover of PIA/PNAG or a repressor of its synthesis, whose expression would be sigmaB-dependent.

Bacterial Proteins↗
PubMed2002

Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose.

We report here a new screening method based on the fluorescence of colonies on calcofluor agar plates to identify transposon insertion mutants of Salmonella enteritidis that are defective in biofilm development. The results not only confirmed the requirement of genes already described for the modulation of multicellular behaviour in Salmonella typhimurium and other species, but also revealed new aspects of the biofilm formation process, such as two new genetic elements, named as bcsABZC and bcsEFG operons, required for the synthesis of an exopolysaccharide, digestible with cellulase. Non-polar mutations of bcsC and bcsE genes and complementation experiments demonstrated that both operons are responsible for cellulose biosynthesis in both S. enteritidis and S. typhimurium. Using two different growth media, ATM and LB, we showed that the biofilm produced by S. enteritidis is made of different constituents, suggesting that biofilm composition and regulation depends on environmental conditions. Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose-deficient mutants indicated that, at least under our experimental conditions, the production of cellulose is not involved in the virulence of S. enteritidis. However, cellulose-deficient mutants were more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of S. enteritidis on surface environments.

Animals↗